1. Field of the Invention
The present invention relates to novel, improved retroviral vectors which can be used for gene therapy, more specifically, to retroviral vectors which are not only safer, more versatile, and more convenient than any other existing vectors, but they also drive high levels of gene expression and high viral titer.
2. Description of the Prior Art
Murine leukemia virus(MLV)-based retroviral vectors which are the most widely used gene delivery vehicles in gene therapy clinical trials, have been employed in almost 70% of approved protocols(see: Ali, M. et al., Gene Ther., 1:367-384, 1994; Marshall, E., Science, 269:1050-1055, 1995).
However, despite their frequent use for gene transfer, many of the biochemical and genetic properties of MLV such as cis and trans factors important for gene expression, viral assembly and packing, have not been completely understood. Moreover, there are many problems with the retroviral vectors currently in clinical use such as MFG(see: Bowtell, D. et al., J. Virol., 62:2464-2473, 1988; Ohashi, T. S. et al., Proc. Natl. Acad. Sci., USA, 89:11332-11336, 1992; Jaffe, E. M., Cancer Res., 53:2221-2226, 1993; Bender, M. A. et al., J. Virol., 61:1639-1646, 1987), and LN-based vectors (see: Boggs, S. S. et al., Gene Ther., 2:632-638,1995; Armentano, D. et al., J. Virol., 61:1647-1650, 1987; Adam M. A., and A. D. Miller, J. Virol., 62:3802-3806, 19B8; Osborne, W. R. A., and A. D. Miller, Proc. Natl. Acad. Sci., USA, 85:6851-6855, 1988; Miller, A. D., and Roseman, Biotechniques, 7:980-990, 1989; Palmer, T. D. et al., Blood, 73:438-445, 1989).
First, all retroviral vectors contain sequences which are also present in the packaging lines. Accordingly, recombination between the packaging genome and the vector results in the generation of replication-competent retrovirus(RCR).
Secondly, most retroviral vectors use either long terminal repeat(LTR) sequences from MLV or related LTR sequences such as myeloid proliferation stimulating virus(MPSV), murine sarcoma virus(MSV) or an heterologous internal promoter. Although the LTR works efficiently in certain cell types, its activity can be down-regulated and its presence can affect expression from internal promoters(see: Bowtell, D. D. L. et al., J. Virol., 62:2464-2473, 1988; Emerman, M. and H. M. Temin, Cell, 39:449-467, 1984).
Thirdly, the viral titers achieved with the vectors in current packaging lines, although they are improved ones, are still not sufficiently high enough for many therapeutic applications.
Fourthly, MLV-based vectors packaged in murine packaging lines, are susceptible to complement-mediated inactivation in vivo, which, in turn, limits their utility for in vivo applications(see: Takeuchi, Y. et al., J. Virol., 68:8001-8007, 1994; Cosset F. L. et al., J. Virol., 69:7430-7436, 1995).
Fifthly, it is difficult to produce a virus at a reasonable titer for targeting a specific cell type or tissue by direct, in vivo delivery(see: Kasahara, N. et al., Science, 266:1373-1376, 1994; Kabat D., Science, 269:417, 1995).
Finally, MLV-based vectors, when packaged in murine packaging lines, cannot deliver a gene of interest to non-dividing cells.
Under the circumstances, WO 92/07943 discloses a retroviral vector MFG including an insertion site for genes of interest which are capable of expressing high levels of the protein derived from the genes of interest in a wide variety of transfected cell types, and those lacking a selective marker, thus rendering them suitable for human gene therapy in the treatment of a variety of disease states without the co-expresssing of a selective marker.
In this connection, the present inventors compared the levels of gene expression from several types of retroviral vectors currently used in clinical trials for gene therapy(see: Byun, J. et al., Gene Ther., 3:780-788, 1996). As a result, it has been suggested that the MFG retroviral vector is superior in conferring gene expression after transduction of a variety of target cells, whose results are consistent with the previous reports in the art(see: Ohashi, T. S. et al., Proc. Natl. Acad. Sci., USA, 89:11332-11336, 1992; Riviere I. et al., Proc. Natl. Acad. Sci., USA, 92:6733-6737, 1995; Krall, W. J. et al., Gene Therapy, 3:37-48, 1996).
However, it has been found that the MFG retroviral vector has many features that should be modified in terms of gene expression, viral titers and safety as follows:
First, MFG contains significantly long coding sequences for gag and env used as a template for homologous recombination, which increase the possibility of the generation of replication-competent retrovirus (RCR).
Secondly, MFG-mediated gene expression is driven by the MLV LTR which is a medium-strength constitutive promoter. However, for many therapeutic applications, it would be necessary to regulate gene expression, not only in terms of levels but also timing of expression, in a sophisticated manner. Accordingly, if heterologous promoter elements are inserted to U3 or U3 is replaced with other full-size promoters, the retroviral vector can be applied in a wide variety of gene therapy. In this connection, the enhancer region of MLV U3 is replaced with heterologous enhancers and U3 is substituted with U3 from similar retroviruses such as MPSV and Friend MLV, nevertheless, it is not clear whether U3 can be replaced with a completely different sequence.
Thirdly, the original version of MFG is designed for expression of a single gene, though the expression of more than two or three genes is desirable for many therapeutic applications.
Finally, NcoI which coincides with the ATG codon of the env gene should be the expression site in MFG to obtain high levels of gene expression, which, in turn, restricts the broader use of MFG retroviral vector.
Naturally, in order for retroviral vectors including MFG to be clinically viable forms of gene delivery, some or all of the current limitations have to be addressed. Accordingly, there are strong reasons for exploring novel and improved retroviral vectors which are capable of expressing controlled levels of proteins derived from the genes of interest in a wide range of transfected cells.